|
| Status |
Public on May 10, 2016 |
| Title |
PHH_WO_LD_exp3 [miRNA] |
| Sample type |
RNA |
| |
|
| Source name |
PHH_WO_LD_exp3
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: primary human hepatocytes
main source: liver
study type: Wash out effect
assay type: ex vivo
strain: Hu4227, Hu8150, Hu4197
agent: aflatoxin B1
dose: 0.3
dose unit: uM
dose duration: 5
duration unit: days
dose frequency: daily
vehicle: DMSO
route: medium
|
| Treatment protocol |
After quick thawing in a water bath at 37°C, viability of the cells was checked by a Trypan blue (CAS no. 72-57-1, Sigma–Aldrich) exclusion test as instructed in the supplier’s protocol (Invitrogen, 2012). All viability scores after thawing were in agreement with those listed by the supplier. Before AFB1 treatment, cells were allowed to acclimatize for 48 hours. This is needed for the hepatocytes to restore an in vivo like cellular configuration and enzyme expression levels as optimally as possible.AFB1 doses causing minimal (IC20) cytotoxicity after 5 days of repetitive daily exposure were established by means of the MTT assay (23). Furthermore, crucial liver function enzymes such as lactate dehydrogenase (LDH) and alanine transaminase (ALT) were measured. LDH and ALT were spectrophotometrically determined on a Cobas 8000 Modular Analyser (Roche Diagnostics, Basel, Switzerland). Based on this data, 0.3 µM of AFB1, was selected for the main experiment.
|
| Growth protocol |
Cryopreserved primary human hepatocytes (PHH) were purchased from Life Technologies. Cells were cultured in pre-coated 24-well and 6-well plates (700,000 cells/ml) in a 2-layer collagen sandwich (A11428-02, Gibco), according to the supplier’s protocol (Invitrogen, 2012). The following culture media were used: Hepatocyte Thawing Medium (HTM) for thawing (CM7500, Gibco), Williams’ Medium E (1x, no phenol red) (A1217601, Gibco) + Cell Maintenance supplement B kit (CM4000, Gibco) for plating and incubation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were cultured in triplicate in 24 wells (RNA) or 6 wells (DNA) in a collagen sandwich layer. Following 5 days of repetitive daily exposure the treatment was removed for 3 days (washout period), then cells lysates were harvested for DNA and total RNA isolation. At the end of washout, medium was removed and PHH were harvested in Qiazol (Qiagen). Total RNA was isolated using a miRNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol and followed by DNase I (Qiagen) treatment. Upon purification, RNA concentrations were measured by means of a NanoDrop® ND-1000 spectrophotometer (Thermo Scientific) at 260 and 280 nm. RNA quality and integrity were assessed by using automated gel electrophoresis on an Agilent 2100 Bioanalyzer system (Agilent Technologies). Only RNA samples which showed clear 18S and 28S peaks and with an RNA integrity number (RIN) higher than 8, were used. Samples were stored at -80ºC until RNA hybridization.
|
| Label |
Cy3
|
| Label protocol |
MicroRNA expression profiling was performed using Agilent Sureprint G3 Unrestricted Human miRNA V19 8 × 60 K microarrays.
|
| |
|
| Hybridization protocol |
The hybridization was performed following standard protocols, after which the microarray slides were washed and scanned using a DNA microarray scanner (Agilent Technologies).
|
| Scan protocol |
Samples were labeled according to the manufacturers protocol.
|
| Description |
Agilent Sureprint G3 Unrestricted Human miRNA V19 8 × 60 K microarrays
|
| Data processing |
The scanned images were converted into TXT files using the Feature Extraction Software v10.7.3.1 from Agilent Technologies, which were imported in R 2.15.3 (http://www.r-project.org) for quality control with an in-house developed pipeline. Filtering and normalization was performed using AgiMicroRna (32). Total gene signals were log2-transformed and quantile-normalized. The selection criteria for determining differentially expressed microRNAs (DE-miRs) were identical to those for DEGs.
|
| |
|
| Submission date |
Jul 30, 2015 |
| Last update date |
May 10, 2016 |
| Contact name |
Linda Rieswijk |
| E-mail(s) |
linda.rieswijk@maastrichtuniversity.nl
|
| Organization name |
Maastricht University
|
| Department |
Toxicogenomics
|
| Street address |
Universiteitssingel 50
|
| City |
Maastricht |
| ZIP/Postal code |
6229 ER |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL18402 |
| Series (2) |
| GSE71545 |
Aflatoxin B1 induces persistent epigenomic effects in primary human hepatocytes associated with hepatocellular carcinoma [miRNA] |
| GSE71549 |
Aflatoxin B1 induces persistent epigenomic effects in primary human hepatocytes associated with hepatocellular carcinoma |
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